Down-regulation of central glucocorticoid receptor expression using antisense oligonucleotide technology

Endogenous glucocorticoids and serotonin have been implicated in the pathophysiology of depression, anxiety and schizophrenia. This thesis investigates the potential of downregulating expression of central Type II glucocorticoid receptors (GR) both in vitro and in vivo, with empirically-designed antisense oligodeoxynucleotides (ODN), to characterise GR modulation of 5-HT2A receptor expression using quantitative RT-PCR, Western blot analysis and radioligand binding. The functional consequence of GR downregulation is also determined by measuring 1-(2,5-dimethoxy 4-iodophenyl)-2-amino propane hydrochloride (DOI) mediated 5-HT2A receptor specific headshakes. Using a library of random antisense ODN probes, RNAse H accessibility mapping of T7-primed, in vitro transcribed GR mRNA revealed several potential cleavage sites and identified an optimally effect GR antisense ODN sequence of 21-mer length (GRAS5). In vitro efficacy studies using rat C6 glioma cells showed a 56% downregulation in GR mRNA levels and 80% downregulation in GR protein levels. In the same cells a 29% upregulation in 5-HT2A mRNA levels and 32% upregulation in 5-HT2A protein levels was revealed. This confirmed the optimal nature of the GRAS5 sequence to produce marked inhibition of GR gene expression, and also revealed GR modulation of the 50-HT2A receptor subtype in C6 glioma cells to be a tonic repression of receptor expression. The distribution of a fluorescently-labelled GRAS5 ODN was detected in diverse areas of the rat brain after single ICV administration, although this fluorescence signal was not sustained over a period of 5 days. However, fluorescently-labelled GRAS5 ODN, when formulated in polymer microspheres, showed diverse distribution in the brain which was maintained for 5 days following a single ICV administration. This produced no apparent neurotoxic effects on rat behaviour and hypothalamic-pituitary-adrenal (HPA) axis homeostasis. Furthermore, a single polymer microsphere injection ICV proved to be an effective means of delivering antisense ODNs and this was adopted for the in vivo efficacy studies. In vivo characterisation of GRAS5 revealed marked downregulation of GR mRNA in rat brain regions such as the frontal cortex (26%), hippocampus (35%), and hypothalamus (39%). Downregulation of GR protein was also revealed in frontal cortex (67%), hippocampus (76%), and hypothalamus (80%). In the same animals upregulation of 5-HT2A mRNA levels was shown in frontal cortex (13%), hippocampus (7%), and hypothalamus (5%) while upregulation in 5-HT2A protein levels was shown in frontal cortex (21 %). This upregulation in 5-HT2A receptor density as a result of antisense-mediated inhibition of GR was further confirmed by a 55% increase in DOl-mediated 5-HT2A receptor specific headshakes. These results demonstrate that GR is involved in tonic inhibitory regulation of 5-HT2A receptor expression and function in vivo, thus providing the potential to control 5-HT2A-linked disorders through corticosteroid manipulation. These experiments have therefore established an antisense approach which can be used to investigate pharmacological characteristics of receptors.

Design, synthesis and biological evaluation of potential antibacterial oligonucleotides

Purine and pyrimidine triplex-forming oligonucleotides (TFOs), as potential antibacterial agents, were designed to bind by Hoogsteen and reverse Hoogsteen hydrogen bonds in a sequence specific manner in the major groove of genomic DNA at specific polypurine sites within the gyrA gene of E. coli and S. pneumoniae. Sequences were prepared by automated synthesis, with purification and characterisation determined by high performance liquid chromatograpy, capillary electrophoresis and mass spectrometry. Triplex stability was assessed using melting curves where the binding of the third strand to the duplex target, was assessed over a temperature range of 0-80°C, and at pH 6.4 and 7.2. The most successful of the unmodified TFOs (6) showed a Tm value of 26 °C at both pH values with binding via reverse Hoogsteen bonds. Binding to genomic DNA was also demonstrated by spectrofluorimetry, using fluorescein-labelled TFOs, from which dissociation constants were determined. Modifications in the form of 5mC, 5' acridine attachment, phosphorothioation, 2'-0-methylation and phosphoramidation, were made in order to. increase Tm values. Phosphoramidate modification was the most with increased Tm values of 42°C. However, the final purity of these sequences was poor due to their difficult syntheses. FACS (fluorescent activated cell sorting) analysis was used to determine the potential uptake of a fluorescently labelled analogue of 6 via passive, coJd shock mediated, and anionic liposome aided, uptake. This was established at 20°C and 37°C. At both temperatures anionic lipid-mediated uptake produced unrivalled fluorescence, equivalent to 20 and 43% at 20 and 37°C respectively. Antibacterial activity of each oligonucleotide was assessed by viable count anaJysis relying on passive uptake, cold shocking techniques, chlorpromazine-mediated uptake, and, cationic and anionic lipid-aided uptake. All oligonucleotides were assessed for their ability to enhance uptake, which is a major barrier to the effectiveness of these agents. Compound 6 under cold shocking conditions produced the greatest consistent decline in colony forming units per ml. Results for this compound were sometimes variable indicating inconsistent uptake by this particular assay method.

Introduction of structural diversity into oligonucleotides containing 6-thioguanine via on-column conjugation

A method is described for the introduction of structural diversity into the thiocarbonyl group of 6-thioguanine within support-bound, fully protected oligonucleotides via 'on-column′ conjugation. 2′-Deoxy-6-thioguanosine with a chemically-labile trigger at its 6-thio function was incorporated at defined sites into chemically synthesized oligonucleotides. Following selective removal of the thio-protection group the support-immobilized oligonucleotides were conjugated with various groups on-column, and then deprotected and purified to produce a number of oligomers each containing a different modified base. Since the modification is accomplished on-column without affecting other functional and protecting groups in the oligomers this method is compatible with introducing structural diversity at multiple sites in DNA. © 2003 Elsevier Science Ltd. All rights reserved.

Selective inhibition of the human tie-1 promoter with triplex-forming oligonucleotides targeted to ets binding sites

The Tie receptors (Tie-1 and Tie-2/Tek) are essential for angiogenesis and vascular remodeling/integrity. Tie receptors are up-regulated in tumor-associated endothelium, and their inhibition disrupts angiogenesis and can prevent tumor growth as a consequence. To investigate the potential of anti-gene approaches to inhibit tie gene expression for anti-angiogenic therapy, we have examined triple-helical (triplex) DNA formation at 2 tandem Ets transcription factor binding motifs (designated E-1 and E-2) in the human tie-1 promoter. Various tie-1 promoter deletion/mutation luciferase reporter constructs were generated and transfected into endothelial cells to examine the relative activities of E-1 and E-2. The binding of antiparallel and parallel (control) purine motif oligonucleotides (21-22 bp) targeted to E-1 and E-2 was assessed by plasmid DNA fragment binding and electrophoretic mobility shift assays. Triplex-forming oligonucleotides were incubated with tie-1 reporter constructs and transfected into endothelial cells to determine their activity. The Ets binding motifs in the E-1 sequence were essential for human tie-1 promoter activity in endothelial cells, whereas the deletion of E-2 had no effect. Antiparallel purine motif oligonucleotides targeted at E-1 or E-2 selectively formed strong triplex DNA (K(d) approximately 10(-7) M) at 37 degrees C. Transfection of tie-1 reporter constructs with triplex DNA at E-1, but not E-2, specifically inhibited tie-1 promoter activity by up to 75% compared with control oligonucleotides in endothelial cells. As similar multiple Ets binding sites are important for the regulation of several endothelial-restricted genes, this approach may have broad therapeutic potential for cancer and other pathologies involving endothelial proliferation/dysfunction.

Gene therapy tools: oligonucleotides and peptides

Genetic mutations can cause a wide range of diseases, e.g. cancer. Gene therapy has the potential to alleviate or even cure these diseases. One of the many gene therapies developed so far is RNA-cleaving deoxyribozymes, short DNA oligonucleotides that specifically bind to and cleave RNA. Since the development of these synthetic catalytic oligonucleotides, the main way of determining their cleavage kinetics has been through the use of a laborious and error prone gel assay to quantify substrate and product at different time-points. We have developed two new methods for this purpose. The first one includes a fluorescent intercalating dye, PicoGreen, which has an increased fluorescence upon binding double-stranded oligonucleotides; during the course of the reaction the fluorescence intensity will decrease as the RNA is cleaved and dissociates from the deoxyribozyme. A second method was developed based on the common denominator of all nucleases, each cleavage event exposes a single phosphate of the oligonucleotide phosphate backbone; the exposed phosphate can simultaneously be released by a phosphatase and directly quantified by a fluorescent phosphate sensor. This method allows for multiple turnover kinetics of diverse types of nucleases, including deoxyribozymes and protein nucleases. The main challenge of gene therapy is often the delivery into the cell. To bypass cellular defenses researchers have used a vast number of methods; one of these are cell-penetrating peptides which can be either covalently coupled to or non-covalently complexed with a cargo to deliver it into a cell. To further evolve cell-penetrating peptides and understand how they work we developed an assay to be able to quickly screen different conditions in a high-throughput manner. A luciferase up- and downregulation experiment was used together with a reduction of the experimental time by 1 day, upscaling from 24- to 96-well plates and the cost was reduced by 95% compared to commercially available assays. In the last paper we evaluated if cell-penetrating peptides could be used to improve the uptake of an LNA oligonucleotide mimic of GRN163L, a telomerase-inhibiting oligonucleotide. The combination of cell-penetrating peptides and our mimic oligonucleotide lead to an IC50 more than 20 times lower than that of GRN163L.

Simulating the aggregation of DNA oligonucleotides

In this work we have studied, by means of Molecular Dynamics simulations, the process of denaturation and self-assembly of short oligonucleotides. Supramolecular ordering of DNA short strands is a promising field which is constantly enriched with new findings. Examples are provided by micellar and fibrils formations and due to the selectivity of DNA bindings, "intelligent" devices have been developed to perform simple logic operations. It is worth to notice that computer simulations of these DNA nanosystems would complement experiments with detailed insight into processes involved in self-assembly. In order to obtain an accurate description of the interactions involved in the complex structure of DNA we used oxDNA, a coarse-grained model developed by Ouldridge. We simulated the melting transition of 4, 6, and 8 base pair sequences. Sequence and length dependence were analyzed, specifically we compared thermodynamic parameters DeltaH, DeltaS and the melting temperature with literature results. Moreover, we have attempted to reproduce liquid crystal ordering of the ultrashort sequence GCCG at relatively high saline concentration, until now only experimentally observed in Bellini's works. We found that our simple model successfully reproduces the experimental phase sequence (isotropic, nematic, columnar) at T= 5 °C as a function of oligonucleotide concentration, and we fully characterized the microscopic structure of the three phases.

The Analgesic Effect Of Dipyrone In Peripheral Tissue Involves Two Different Mechanisms: Neuronal K(atp) Channel Opening And Cb(1) Receptor Activation.

Dipyrone (metamizole) is an analgesic pro-drug used to control moderate pain. It is metabolized in two major bioactive metabolites: 4-methylaminoantipyrine (4-MAA) and 4-aminoantipyrine (4-AA). The aim of this study was to investigate the participation of peripheral CB1 and CB2 cannabinoid receptors activation in the anti-hyperalgesic effect of dipyrone, 4-MAA or 4-AA. PGE2 (100ng/50µL/paw) was locally administered in the hindpaw of male Wistar rats, and the mechanical nociceptive threshold was quantified by electronic von Frey test, before and 3h after its injection. Dipyrone, 4-MAA or 4-AA was administered 30min before the von Frey test. The selective CB1 receptor antagonist AM251, CB2 receptor antagonist AM630, cGMP inhibitor ODQ or KATP channel blocker glibenclamide were administered 30min before dipyrone, 4-MAA or 4-AA. The antisense-ODN against CB1 receptor expression was intrathecally administered once a day during four consecutive days. PGE2-induced mechanical hyperalgesia was inhibited by dipyrone, 4-MAA, and 4-AA in a dose-response manner. AM251 or ODN anti-sense against neuronal CB1 receptor, but not AM630, reversed the anti-hyperalgesic effect mediated by 4-AA, but not by dipyrone or 4-MAA. On the other hand, the anti-hyperalgesic effect of dipyrone or 4-MAA was reversed by glibenclamide or ODQ. These results suggest that the activation of neuronal CB1, but not CB2 receptor, in peripheral tissue is involved in the anti-hyperalgesic effect of 4-aminoantipyrine. In addition, 4-methylaminoantipyrine mediates the anti-hyperalgesic effect by cGMP activation and KATP opening.

Inflammatory Sensitization Of Nociceptors Depends On Activation Of Nmda Receptors In Drg Satellite Cells.

The present study evaluated the role of N-methyl-D-aspartate receptors (NMDARs) expressed in the dorsal root ganglia (DRG) in the inflammatory sensitization of peripheral nociceptor terminals to mechanical stimulation. Injection of NMDA into the fifth lumbar (L5)-DRG induced hyperalgesia in the rat hind paw with a profile similar to that of intraplantar injection of prostaglandin E2 (PGE2), which was significantly attenuated by injection of the NMDAR antagonist D(-)-2-amino-5-phosphonopentanoic acid (D-AP-5) in the L5-DRG. Moreover, blockade of DRG AMPA receptors by the antagonist 6,7-dinitroquinoxaline-2,3-dione had no effect in the PGE2-induced hyperalgesia in the paw, showing specific involvement of NMDARs in this modulatory effect and suggesting that activation of NMDAR in the DRG plays an important role in the peripheral inflammatory hyperalgesia. In following experiments we observed attenuation of PGE2-induced hyperalgesia in the paw by the knockdown of NMDAR subunits NR1, NR2B, NR2D, and NR3A with antisense-oligodeoxynucleotide treatment in the DRG. Also, in vitro experiments showed that the NMDA-induced sensitization of cultured DRG neurons depends on satellite cell activation and on those same NMDAR subunits, suggesting their importance for the PGE2-induced hyperalgesia. In addition, fluorescent calcium imaging experiments in cultures of DRG cells showed induction of calcium transients by glutamate or NMDA only in satellite cells, but not in neurons. Together, the present results suggest that the mechanical inflammatory nociceptor sensitization is dependent on glutamate release at the DRG and subsequent NMDAR activation in satellite glial cells, supporting the idea that the peripheral hyperalgesia is an event modulated by a glutamatergic system in the DRG.

An oligonucleotide primer set for PCR amplification of the complete honey bee mitochondrial genome

Mitochondrial DNA markers have been widely used to address population and evolutionary questions in the honey bee Apis mellifera. Most of the polymorphic markers are restricted to few mitochondrial regions. Here we describe a set of 24 oligonucleotides that allow PCR amplification of the entire mitochondrial genome of the honey bee A. mellifera in 12 amplicons. These fragments have important applications for the study of mitochondrial genes in different subspecies of A. mellifera and as heterospecific probes to characterize mitochondrial genomes in other bee species.

Characterization of global transcription profile of normal and HPV-immortalized keratinocytes and their response to TNF treatment

Background: Persistent infection by high risk HPV types (e.g. HPV-16, -18, -31, and -45) is the main risk factor for development of cervical intraepithelial neoplasia and cervical cancer. Tumor necrosis factor (TNF) is a key mediator of epithelial cell inflammatory response and exerts a potent cytostatic effect on normal or HPV16, but not on HPV18 immortalized keratinocytes. Moreover, several cervical carcinoma-derived cell lines are resistant to TNF anti-proliferative effect suggesting that the acquisition of TNF-resistance may constitute an important step in HPV-mediated carcinogenesis. In the present study, we compared the gene expression profiles of normal and HPV16 or 18 immortalized human keratinocytes before and after treatment with TNF for 3 or 60 hours. Methods: In this study, we determined the transcriptional changes 3 and 60 hours after TNF treatment of normal, HPV16 and HPV18 immortalized keratinocytes by microarray analysis. The expression pattern of two genes observed by microarray was confirmed by Northern Blot. NF-kappa B activation was also determined by electrophoretic mobility shift assay (EMSA) using specific oligonucleotides and nuclear protein extracts. Results: We observed the differential expression of a common set of genes in two TNF-sensitive cell lines that differs from those modulated in TNF-resistant ones. This information was used to define genes whose differential expression could be associated with the differential response to TNF, such as: KLK7 (kallikrein 7), SOD2 (superoxide dismutase 2), 100P (S100 calcium binding protein P), PI3 (protease inhibitor 3, skin-derived), CSTA (cystatin A), RARRES1 (retinoic acid receptor responder 1), and LXN (latexin). The differential expression of the KLK7 and SOD2 transcripts was confirmed by Northern blot. Moreover, we observed that SOD2 expression correlates with the differential NF-kappa B activation exhibited by TNF-sensitive and TNF-resistant cells. Conclusion: This is the first in depth analysis of the differential effect of TNF on normal and HPV16 or HPV18 immortalized keratinocytes. Our findings may be useful for the identification of genes involved in TNF resistance acquisition and candidate genes which deregulated expression may be associated with cervical disease establishment and/or progression.

Identification of protein-coding and non-coding RNA expression profiles in CD34(+) and in stromal cells in refractory anemia with ringed sideroblasts

Background: Myelodysplastic syndromes (MDS) are a group of clonal hematological disorders characterized by ineffective hematopoiesis with morphological evidence of marrow cell dysplasia resulting in peripheral blood cytopenia. Microarray technology has permitted a refined high-throughput mapping of the transcriptional activity in the human genome. Non-coding RNAs (ncRNAs) transcribed from intronic regions of genes are involved in a number of processes related to post-transcriptional control of gene expression, and in the regulation of exon-skipping and intron retention. Characterization of ncRNAs in progenitor cells and stromal cells of MDS patients could be strategic for understanding gene expression regulation in this disease. Methods: In this study, gene expression profiles of CD34(+) cells of 4 patients with MDS of refractory anemia with ringed sideroblasts (RARS) subgroup and stromal cells of 3 patients with MDS-RARS were compared with healthy individuals using 44 k combined intron-exon oligoarrays, which included probes for exons of protein-coding genes, and for non-coding RNAs transcribed from intronic regions in either the sense or antisense strands. Real-time RT-PCR was performed to confirm the expression levels of selected transcripts. Results: In CD34(+) cells of MDS-RARS patients, 216 genes were significantly differentially expressed (q-value <= 0.01) in comparison to healthy individuals, of which 65 (30%) were non-coding transcripts. In stromal cells of MDS-RARS, 12 genes were significantly differentially expressed (q-value <= 0.05) in comparison to healthy individuals, of which 3 (25%) were non-coding transcripts. Conclusions: These results demonstrated, for the first time, the differential ncRNA expression profile between MDS-RARS and healthy individuals, in CD34(+) cells and stromal cells, suggesting that ncRNAs may play an important role during the development of myelodysplastic syndromes.

Synthesis of selenium- and tellurium-containing nucleosides derived from uridine

The synthesis of selenium- and tellurium-containing nucleosides, derived from uridine is described herein. These compounds were prepared in a concise and short synthetic route in good yields, by nucleophilic substitution of a tosylate group by organoselenium nucleophiles. (C) 2009 Elsevier Ltd. All rights reserved.

Cdc42p controls yeast-cell shape and virulence of Paracoccidioides brasiliensis

Paracoccidioides brasiliensis is characterized by a multiple budding phenotype and a polymorphic cell growth, leading to the formation of cells with extreme variations in shape and size. Since Cdc42 is a pivotal molecule in establishing and maintaining polarized growth for diverse cell types, as well as during pathogenesis of certain fungi, we evaluated its role during cell growth and virulence of the yeast-form of P. brasiliensis. We used antisense technology to knock-down PbCDC42`s expression in P. brasiliensis yeast cells, promoting a decrease in cell size and more homogenous cell growth, altering the typical polymorphism of wild-type cells. Reduced expression levels also lead to increased phagocytosis and decreased virulence in a mouse model of infection. We provide genetic evidences underlying Pbcdc42p as an important protein during host-pathogen interaction and the relevance of the polymorphic nature and cell size in the pathogenesis of P. brasiliensis. (C) 2009 Elsevier Inc. All rights reserved.

Mipomersen, an apolipoprotein B synthesis inhibitor, for lowering of LDL cholesterol concentrations in patients with homozygous familial hypercholesterolaemia: a randomised, double-blind, placebo-controlled trial

Background Homozygous familial hypercholesterolaemia is a rare genetic disorder in which both LDL-receptor alleles are defective, resulting in very high concentrations of LDL cholesterol in plasma and premature coronary artery disease. This study investigated whether an antisense inhibitor of apolipoprotein B synthesis, mipomersen, is effective and safe as an adjunctive agent to lower LDL cholesterol concentrations in patients with this disease. Methods This randomised, double-blind, placebo-controlled, phase 3 study was undertaken in nine lipid clinics in seven countries. Patients aged 12 years and older with clinical diagnosis or genetic confirmation of homozygous familial hypercholesterolaemia, who were already receiving the maximum tolerated dose of a lipid-lowering drug, were randomly assigned to mipomersen 200 mg subcutaneously every week or placebo for 26 weeks. Randomisation was computer generated and stratified by weight (<50 kg vs >= 50 kg) in a centralised blocked randomisation, implemented with a computerised interactive voice response system. All clinical, medical, and pharmacy personnel, and patients were masked to treatment allocation. The primary endpoint was percentage change in LDL cholesterol concentration from baseline. Analysis was by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT00607373. Findings 34 patients were assigned to mipomersen and 17 to placebo; data for all patients were analysed. 45 patients completed the 26-week treatment period (28 mipomersen, 17 placebo). Mean concentrations of LDL cholesterol at baseline were 11.4 mmol/L (SD 3.6) in the mipomersen group and 10.4 mmol/L (3.7) in the placebo group. The mean percentage change in LDL cholesterol concentration was significantly greater with mipomersen (-24.7%, 95% CI 31.6 to 17.7) than with placebo (-3.3%, 12.1 to 5.5; p=0.0003). The most common adverse events were injection-site reactions (26 [76%] patients in mipomersen group vs four [24%] in placebo group). Four (12%) patients in the mipomersen group but none in the placebo group had increases in concentrations of alanine aminotransferase of three times or more the upper limit of normal. Interpretation Inhibition of apolipoprotein B synthesis by mipomersen represents a novel, effective therapy to reduce LDL cholesterol concentrations in patients with homozygous familial hypercholesterolaemia who are already receiving lipid-lowering drugs, including high-dose statins.